Skin color is determined by a combination of the pigments produced in the skin and natural colors of the upper layers of the skin. The epidermis, which is the outer epithelial layer of the skin, contains epidermal melanocytes which produce the skin pigmentation, referred to as melanin. In patients with hypopigmentation or depigmentation of the skin, the melanocytes in the skin are absent, destroyed or non-functional. As a result, white patches of skin appear on different parts of the body.
Pigmentation is not only a protective function of the melanocytes, but also plays an important role in cutaneous aesthetics. Hypopigmentation or depigmentation of the skin results from specific disorders or conditions affecting the pigmentation system through either local destruction or absence of melanocytes or through inhibition of their function. Although this situation is clinically benign, the psychological and social consequences can be debilitating.
Vitiligo is a specific skin pigment disorder characterized by the destruction of epidermal melanocytes and development of patchy depigmented lesions. Current treatments, which include the use of photosensitizes (e.g. psoralens) with UVA radiation (PUVA) or topical corticosteroids, have low success rates and are generally accompanied by unpleasant side effects (Shaffali and Gawkrodger, 2000, Clin Exp Dermatol 25:575).
Surgical skin grafting techniques have been applied to patients with vitiligo, including transplantation of blister tops or minigrafts from normal skin (Koga et al., 1988, Arch Dermatol. 124:1656; Gupta et al., 1999, Dermatol Surg 25:955; Falabella, 1984, J Dermatol Surg. Oncol. 10:136; Suvanprakorn et al., 1985, J Am Acad Derm 13:968; Sarkar et al., 2001, J Dermatol 28:540; Pai et al., 2002 J. Eur Acad Dermatol Venereol 16:604). These treatments have been successful where the area of the lesion is small and localized. However, in individuals who have either large depigmented patches or many depigmented patches, it is difficult to obtain enough graft to cover all the lesions (Van Geel, N. et al., 2001, Dermatology 202:162).
In order to achieve success in autologous skin transplants for large areas of the skin, it is often necessary to obtain skin grafts with large number of cells, in particular melanocytes. Alternatively, one can expand a population of autologous melanocytes in culture for transplantation. This approach entails obtaining a population of melanocytes from the patient, culturing this population of cells in vitro under conditions that promote cell proliferation, cell migration and production of melanogenesis, and reintroducing the cells to the patient's skin under conditions that promote repigmentation.
A number of different factors have been found to stimulate the growth of melanocytes in culture. For example, hepatocyte growth factor (HGF) is a pleiotrophic growth factor family initially identified as a potent mitogen for cultured hepatocytes. HGF has been shown to stimulate growth and migration of various epithelial cells and vascular endothelial cells (Matsumoto et al., 1996, J. Biochem. 119:591; Jiang et al., 1997, Histol Histopathol 12:537). In addition, it has been shown that HGF stimulates growth of cultured ocular melanocytes (Hu, 2000, Pigment Cell Res 13 (Suppl. 8): 81) and in skin, HGF can be produced by the fibroblasts in the dermis (Halaban et al., 1993, Ann New York Acad Sci 680:290).
α-Stimulating Factor (αMSH) is a tridecapeptide with a sequence identical to the first 13 amino acids of adrenocorticotropin (ACTH). Keratinocytes appear to be a major source of the peptide (Thody et al., 1998, Pigment Cell Res 11:265; Wakamatsu et al., 1997, Pigment Cell Res 10:288). Previous reports on the effect of αMSH on cultured epidermal melanocytes have been conflicting (Halaban et al., 1988, J Cell Biol 107:1611; Thody and Graham., 1998, Pigment Cell Res 11:265; Wakamatsu et al., 1997, Pigment Cell Res 10:288; Abdel-Malek et al., 1995, Proc. Natl. Acad. Sci USA 92:1789). For example, Halaban et al., reported that αMSH failed to stimulate growth on cultured epidermal melanocytes (Halaban et al., 1988, J Cell Bol 107:1611). However, the lack of a response in these studies may be related to the addition of cholera toxin to the media.
Epinephrine is a potent stimulator of both α- and β-adrenergic receptors. Epinephrine activates the β2-adrenergic receptors through the cAMP second messenger system to stimulate melanocytes. β2-adrenergic agonists stimulate growth and melanogenesis of ocular melanocytes and α-, β1- and β3-adrenergic agonists did not have any effect on growth or melanogenesis (Hu et al., 2000, Exp. Eye Res. 71:217).
Lerner et al. (1987, J Invest Derm 89:219) described the use of melanocyte transplantation for the treatment of piebaldism, a genetic disorder characterized by congenital patches of white skin and hair that lack melanocytes. A small piece of skin was obtained from the patient by shave biopsy and the epidermal melanocytes were cultured using a culture medium containing TPA (12-O-tetradecanoyl-phorbol-13-acetate), IBMX (3-isobutyl-1-methylxanthine), cholera toxin and newborn calf serum. Though cells were successfully transplanted to an area of hypopigmentation, the use of TPA, a well-known tumor promoting factor, in the medium discourages its applications in human transplantations.
U.S. Pat. No. 4,757,019 also discloses the use of a culture medium for culturing human melanocytes. This medium comprises MEM, a basal medium, with 5% fetal bovine serum, phorbol 12-myristate13-acetate (PMA, also known as TPA 10 ng/ml) and cholera toxin (10−8 M). Since this medium also contains toxic compounds, i.e., PMA and cholera toxin it would not be appropriate for applications in human transplantation.
Olsson and Juhlin (1992, Lancet 340:981) reported the use of an epidermal melanocyte culture medium which was a formulation containing PC-1, supplemented with basic fibroblast growth factor (bFGF), dbcAMP, penicillin and streptomycin. The culture medium was used to expand isolated epidermal melanocytes for use in autologous melanocyte transplantation. This medium did not contain any serum, which is necessary for the growth and differentiation of epidermal melanocytes. In addition, the concentration of bFGF (5 ng/ml) in the medium was too low to stimulate adequate growth of epidermal melanocytes. Furthermore, the dbcAMP added to the medium is not a natural substance and only acts for a short period of time. In addition, no quantitative data on the number of cellular divisions and melanin content of the expounded cells have been reported, therefore, it is difficult to evaluate the efficiency of this medium. It is noteworthy, however, that Olsson and Juhlin later described the use of a non-cultured epidermal melanocyte transplantation method for the treatment of vitiligo (Olsson and Juhlin, 1998, Br J Dermatol 138:644).
Additional culture media, e.g., HU16 (FIC medium) has been developed for culturing epidermal melanocytes. HU16 medium comprises F12 medium, a commercially available basal medium, supplemented with an optimal concentration of bFGF, 3-isobutyl-1-methxanthine (IBMX), cholera toxin and fetal bovine serum. This medium has been used successfully to grow epidermal melanocytes for melanocyte transplantation treatment of vitiligo in over 120 patients in Taiwan. (Chen et al., 1999, Show Chwan Med J 1:85; Chen et al., 2000, J. Dermatol 27:434; Chen et al., 2001, J Am Acad Dermatol 44:545)
Although the HU16 medium stimulated a great increase in melanocyte growth, the disadvantages of this medium include that (i) the IBMX and cholera toxin contained in this medium are non-physiological, non-natural substances and cholera toxin has negative psychological implications for physicians and patients, and (ii) the growth and melanogenesis stimulating effects of this medium, although better than for any previously reported media, is not optimal. Thus, some patients may wait for prolonged periods of time for the cells to grow and a small percentage of patient's cells fail to ever grow well enough to meet the requirement of transplantation. Moreover, even with a successful transplant, some patients have a lack of pigmentation at the periphery of the transplanted region, possibly due to insufficient stimulation of cell migration provided by this medium (Chen et al. 1999, Show Chwan Med J 1999 1:85; Chen et al., 2000, J. Dermatol 27:434).
The requirements for medium useful for clinical cell transplantation are different from those of regular culture medium. For example, medium for clinical cell transplantation should contain all natural, physiological substances, so that it will not damage the cell or pose a risk of harm to the patient after cell transplantation. Furthermore, the culture medium should provide for cell growth, melanogenesis and migration. None of the previously reported culture media use for epidermal melanocyte transplantation has been found to meet these criteria, nor have quantitative tests of the effects of all these culture media on cell growth, melanogenesis and migration have not been reported.
Therefore, there is a need to develop novel compositions comprising non-toxic and/or natural and/or physiologically compatible components and methods of culturing epidermal melanocytes in vitro under conditions that promote epidermal melanocyte proliferation, as well as stimulate normal epidermal melanocyte cell properties, including melanin production and cell migration.